Journal: International Journal of Medical Sciences

Article Title: SSRP1 influences colorectal cancer cell growth and apoptosis via the AKT pathway

doi: 10.7150/ijms.38439

Figure Lengend Snippet: SSRP1 exerts proliferation and apoptosis of colorectal cancer cells by activating the AKT pathway. (A) Graphs with quantitative data for the flow cytometric cell cycle distribution assay in HCT15 and SW620 cells. (B, C) FITC Annexin V/PI staining indicated increased apoptosis in HCT15 and SW620 cells with SSRP1 inhibiting. (D, E) Western blot displayed reduced P-AKT, CyclinD1, BCL-2 and MDM2, accompanying increased p27, p21, p53 and BAX in transfected HCT15 and SW620 cells. (F, G) Apoptosis ratio in HCT15 cells with SSRP1 or AKT inhibitors through flow cytometry. (H, I) The protein level of P-AKT, AKT, BAX and BCL-2 in HCT15 cells with SSRP1 or AKT inhibitors. * P < 0.05, ** P < 0.01 vs NC group.

Article Snippet: Nonspecific binding sites were blocked with 5% low-fat milk and 0.1% Tween-20 at room temperature for 2 h. Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: Proteintech (Wuhan, China): anti-SSRP1 (1:1000; 15696-1-AP), anti-p27 (1:1000; 25614-1-AP), anti-p21 (1:1000; 10355-1-AP), anti-CyclinD1 (1:1000; 60186-1-Ig), anti-BCL2 (1:1000; 26593-1-AP), anti-BAX (1:1000; 50599-2-Ig), anti-MDM2 (1:1000; 19058-1-AP), anti-p53 (1:1000; 10442-1-AP), anti-AKT (1:1000; 60203-2-Ig), anti-β-actin (1:2000; 66009-1-Ig) and arigo (Taiwan, China): anti-P-AKT (1:1000; ARG51559); secondary antibody: goat anti-Mouse (1:2000; 10828-I-AP) and goat anti-Rabbit IgG (H+L) (1:2000; SA0000I-2) from Proteintech Group Inc.

Techniques: Staining, Western Blot, Transfection, Flow Cytometry

Journal: International Journal of Medical Sciences

Article Title: SSRP1 influences colorectal cancer cell growth and apoptosis via the AKT pathway

doi: 10.7150/ijms.38439

Figure Lengend Snippet: SSRP1 inhibition blocks the growth of colorectal cancer in vivo . (A) Treatment for transplanted xenogeneic models using HCT15 cells. (B) Image of the tumor tissues of NC group and siSSRP1 group. (C) Tumor growth curve of NC group and siSSRP1 group for 34 d treatment. (D) Immunofluorescent staining showing that SSRP1 expression was reduced in siSSRP1 group, compared with NC group. (E) Validation of SSPR1 protein levels through western blot. (F) TUNEL assay of tumor tissue. (G) Immunofluorescent staining of PCNA, BAX, Bcl2, MMP-2 and MMP-9. (H) AKT, P-AKT, p27, p21, CyllinD1, p53, MDM2, BAX, and Bcl2 protein levels were determined by western blot analysis. * P <0.05, ** P <0.01 vs NC group.

Article Snippet: Nonspecific binding sites were blocked with 5% low-fat milk and 0.1% Tween-20 at room temperature for 2 h. Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: Proteintech (Wuhan, China): anti-SSRP1 (1:1000; 15696-1-AP), anti-p27 (1:1000; 25614-1-AP), anti-p21 (1:1000; 10355-1-AP), anti-CyclinD1 (1:1000; 60186-1-Ig), anti-BCL2 (1:1000; 26593-1-AP), anti-BAX (1:1000; 50599-2-Ig), anti-MDM2 (1:1000; 19058-1-AP), anti-p53 (1:1000; 10442-1-AP), anti-AKT (1:1000; 60203-2-Ig), anti-β-actin (1:2000; 66009-1-Ig) and arigo (Taiwan, China): anti-P-AKT (1:1000; ARG51559); secondary antibody: goat anti-Mouse (1:2000; 10828-I-AP) and goat anti-Rabbit IgG (H+L) (1:2000; SA0000I-2) from Proteintech Group Inc.

Techniques: Inhibition, In Vivo, Staining, Expressing, Western Blot, TUNEL Assay

Journal: bioRxiv

Article Title: Replicative stress in gastroesophageal adenocarcinoma is associated with chromosomal instability and sensitivity to DNA damage response inhibitors

doi: 10.1101/2023.03.27.534412

Figure Lengend Snippet: A. Tabulated summary of IHC stainings indicating the number of premalignant (green) and malignant (blue) gastric lesions scored for each staining criterion. DDR/replication stress markers-53BP1, H2AX, pH2AX in red; Cell cycle regulators-p53, p16, p21 in purple. B. Summary of the stainings (DDR markers and the cell cycle regulation markers) in paired premalignant and malignant gastric lesions from gastric cancer patient8. PM= Premalignant gastric lesion, M= Malignant gastric lesion. C. Representative IHC images of paired premalignant and malignant gastric lesions from a gastric cancer patient (patient8) stained for DDR markers and cell cycle regulation markers, including H&E staining. Insets represent the zoomed area for each image.

Article Snippet: The primary antibodies used for immunohistochemistry were: Human tissue-rabbit anti-ki67 (1:1000, Biocare Medical, #CRM325-5b6), rabbit anti-H2AX (1:1000, CST, #7631), rabbit anti-pH2AX ser139 (1:1000, CST, #9718), mouse anti-53BP1 (1:1000, Millipore, #3802), mouse anti-p53 (1:1000, CST, #48818), mouse anti-P16INK4 (1:1000, Abcam, #54210), rabbit anti-p21 (1:1000, CST, #2947), Murine tissue-rabbit anti-ki67 (1:1000, Biocare Medical, #CRM325-5b6), rabbit anti-pH2AX ser139, (1:1000, CST, #9718), rabbit anti-p53 (1:1000, Abcam, #1431), rabbit anti-p21 (1:1000, Abcam, #188224).

Techniques: Staining

Journal: bioRxiv

Article Title: Replicative stress in gastroesophageal adenocarcinoma is associated with chromosomal instability and sensitivity to DNA damage response inhibitors

doi: 10.1101/2023.03.27.534412

Figure Lengend Snippet: A. Summary of dysplasia grade and staining pattern of proliferation marker ki67, DDR marker pH2AX, and cell cycle regulation markers (p53 and p21) in paired normal (grey), premalignant (green), and malignant (blue) gastric lesions of four MNU treated mice. B. Representative IHC images of the staining pattern of proliferation marker ki67, DDR marker pH2AX, and cell cycle regulation markers (p53 and p21) in paired normal, premalignant, and malignant gastric lesions of MNU treated mouse4 (dysplasia score 5).

Article Snippet: The primary antibodies used for immunohistochemistry were: Human tissue-rabbit anti-ki67 (1:1000, Biocare Medical, #CRM325-5b6), rabbit anti-H2AX (1:1000, CST, #7631), rabbit anti-pH2AX ser139 (1:1000, CST, #9718), mouse anti-53BP1 (1:1000, Millipore, #3802), mouse anti-p53 (1:1000, CST, #48818), mouse anti-P16INK4 (1:1000, Abcam, #54210), rabbit anti-p21 (1:1000, CST, #2947), Murine tissue-rabbit anti-ki67 (1:1000, Biocare Medical, #CRM325-5b6), rabbit anti-pH2AX ser139, (1:1000, CST, #9718), rabbit anti-p53 (1:1000, Abcam, #1431), rabbit anti-p21 (1:1000, Abcam, #188224).

Techniques: Staining, Marker

Journal: Carcinogenesis

Article Title: Potent iron chelators increase the mRNA levels of the universal cyclin-dependent kinase inhibitor p21(CIP1/WAF1), but paradoxically inhibit its translation: a potential mechanism of cell cycle dysregulation.

doi: 10.1093/carcin/bgg042

Figure Lengend Snippet: Fig. 3. Western blot analysis demonstrating that DFO or 311 decreased nuclear p21 expression while the DNA-damaging agents Act D and CP increased the expression of this protein in (A) MCF-7 breast cancer cells and (B) MRC-5 fibroblasts. Cells were incubated for 24 h at 37C with control medium (CON) or medium containing DFO (250 mM), 311 (25 mM), Act D (9 nM) or CP (20 mM). Nuclear lysates were then prepared for western blot analysis (see Materials and methods for details). Results from densitometry are tabulated below each figure and are normalized to the b-actin loading control in each case. Results are a typical experiment from four performed.

Article Snippet: The mouse monoclonal antihuman p21 (catalog no. sc-6246; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-human p53 antibodies (catalog no. sc-6243; Santa Cruz Biotechnology) were used at 0.33 and 0.0025 mg/ml, respectively.

Techniques: Western Blot, Expressing, Incubation, Control

Journal: Carcinogenesis

Article Title: Potent iron chelators increase the mRNA levels of the universal cyclin-dependent kinase inhibitor p21(CIP1/WAF1), but paradoxically inhibit its translation: a potential mechanism of cell cycle dysregulation.

doi: 10.1093/carcin/bgg042

Figure Lengend Snippet: Fig. 4. Western blot analysis demonstrating that inhibition of the proteasome using MG132 does not prevent the decrease in nuclear p21 found after treatment with the chelator 311. MCF-7 cells were initially incubated for 24 h at 37C with control medium (CON) or medium containing either DFO (250 mM), 311 (25 mM) or Act D (9 nM). To inhibit proteasomal activity, MG132 (50 mM) was added to the respective treatments and the incubation allowed to continue for a further 3 h at 37C. Nuclear lysates were then prepared for western blot analysis (see Materials and methods for details). Results from densitometry are tabulated below each figure and are normalized to b-actin in each case. Results are a typical experiment from four performed.

Article Snippet: The mouse monoclonal antihuman p21 (catalog no. sc-6246; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-human p53 antibodies (catalog no. sc-6243; Santa Cruz Biotechnology) were used at 0.33 and 0.0025 mg/ml, respectively.

Techniques: Western Blot, Inhibition, Incubation, Control, Activity Assay

Journal: Carcinogenesis

Article Title: Potent iron chelators increase the mRNA levels of the universal cyclin-dependent kinase inhibitor p21(CIP1/WAF1), but paradoxically inhibit its translation: a potential mechanism of cell cycle dysregulation.

doi: 10.1093/carcin/bgg042

Figure Lengend Snippet: Fig. 5. Immunofluorescence studies demonstrating that DFO and 311 do not increase p21 expression but result in nuclear accumulation of p53 in MCF-7 breast cancer cells. Cells were incubated for 24 h at 37C with control medium (CON), DFO (250 mM), 311 (25 mM) or Act D (9 nM) and then prepared for immunofluorescence to assess p21 or p53 distribution (see Materials and methods for details). The magnification used was 100. The exposure period used for photography was equivalent for each picture. Results are a typical experiment from five performed.

Article Snippet: The mouse monoclonal antihuman p21 (catalog no. sc-6246; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-human p53 antibodies (catalog no. sc-6243; Santa Cruz Biotechnology) were used at 0.33 and 0.0025 mg/ml, respectively.

Techniques: Immunofluorescence, Expressing, Incubation, Control

Journal: Carcinogenesis

Article Title: Potent iron chelators increase the mRNA levels of the universal cyclin-dependent kinase inhibitor p21(CIP1/WAF1), but paradoxically inhibit its translation: a potential mechanism of cell cycle dysregulation.

doi: 10.1093/carcin/bgg042

Figure Lengend Snippet: Fig. 6. Western blot analysis demonstrating that 311 prevents nuclear accumulation of p21 in MCF-7 cells treated with the DNA-damaging agent CP, but has no effect on nuclear p53 localization. Cells were incubated for 24 h with either control medium (CON), DFO (250 mM), DFO (250 mM) and CP (20 mM), 311 (25 mM), 311 and CP (20 mM) or CP (20 mM). Nuclear lysates were then prepared for western blot analysis (see Materials and methods for details). Results from densitometry are tabulated below each figure and are normalized to b-actin in each case. Results are a typical experiment from three performed.

Article Snippet: The mouse monoclonal antihuman p21 (catalog no. sc-6246; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-human p53 antibodies (catalog no. sc-6243; Santa Cruz Biotechnology) were used at 0.33 and 0.0025 mg/ml, respectively.

Techniques: Western Blot, Incubation, Control

Journal: Carcinogenesis

Article Title: Potent iron chelators increase the mRNA levels of the universal cyclin-dependent kinase inhibitor p21(CIP1/WAF1), but paradoxically inhibit its translation: a potential mechanism of cell cycle dysregulation.

doi: 10.1093/carcin/bgg042

Figure Lengend Snippet: Fig. 7. Immunofluorescence studies demonstrating that DFO and 311 prevent nuclear accumulation of p21 in MCF-7 cells treated with the DNA-damaging agent Act D, but have no effect on nuclear p53 localization. Cells were incubated for 24 h at 37C with either control medium (CON) or medium containing either DFO (250 mM), DFO (250 mM) and Act D (9 nM), 311 (25 mM), 311 and Act D (9 nM) or Act D (9 nM). After this incubation, cells were then prepared for immunofluorescence to assess p21 or p53 distribution (see Materials and methods for details). The magnification used was 100. The exposure period used for photography was equivalent for each picture. The results are a typical experiment of two performed.

Article Snippet: The mouse monoclonal antihuman p21 (catalog no. sc-6246; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-human p53 antibodies (catalog no. sc-6243; Santa Cruz Biotechnology) were used at 0.33 and 0.0025 mg/ml, respectively.

Techniques: Immunofluorescence, Incubation, Control

Journal: Carcinogenesis

Article Title: Potent iron chelators increase the mRNA levels of the universal cyclin-dependent kinase inhibitor p21(CIP1/WAF1), but paradoxically inhibit its translation: a potential mechanism of cell cycle dysregulation.

doi: 10.1093/carcin/bgg042

Figure Lengend Snippet: Fig. 8. Western blot analysis demonstrating that supplementation of Fe to cells pre-treated with chelators restores nuclear p21 accumulation. MCF-7 cells were initially incubated for 24 h at 37C with either control medium alone (MEM) or 311 (25 mM). This medium was then removed and the cells incubated for a further 24 h at 37C with either MEM, FAC (100 mg/ml), 311 (25 mM) or the Fe complex of 311 (311-Fe) (25 mM). Nuclear lysates were then made for western analysis for p21 and p53, while cytoplasmic lysates were prepared for TfR1 (see Materials and methods for details). Data from densitometry are tabulated below each figure and are normalized to b-actin in each case. Results are a typical experiment from three performed.

Article Snippet: The mouse monoclonal antihuman p21 (catalog no. sc-6246; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-human p53 antibodies (catalog no. sc-6243; Santa Cruz Biotechnology) were used at 0.33 and 0.0025 mg/ml, respectively.

Techniques: Western Blot, Incubation, Control

Journal: Carcinogenesis

Article Title: Potent iron chelators increase the mRNA levels of the universal cyclin-dependent kinase inhibitor p21(CIP1/WAF1), but paradoxically inhibit its translation: a potential mechanism of cell cycle dysregulation.

doi: 10.1093/carcin/bgg042

Figure Lengend Snippet: Fig. 9. Immunofluorescence studies demonstrating that supplementation of iron to cells pre-treated with 311 restores nuclear p21. MCF-7 cells were initially incubated for 24 h at 37C with either control medium alone (MEM), 311 (25 mM) or Act D (9 nM). This medium was then removed and replaced with MEM only or MEM containing the Fe donor FAC (100 mg/ml), 311 (25 mM), the Fe complex of 311 (311-Fe) (25 mM) or Act D (9 nM). After this incubation, cells were then prepared for immunofluorescence to assess p21 distribution (see Materials and methods for details). The magnification used was 100. The exposure period used for photography was equivalent for each picture. Results are a typical experiment from two performed.

Article Snippet: The mouse monoclonal antihuman p21 (catalog no. sc-6246; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-human p53 antibodies (catalog no. sc-6243; Santa Cruz Biotechnology) were used at 0.33 and 0.0025 mg/ml, respectively.

Techniques: Immunofluorescence, Incubation, Control

Journal: Cancer Research

Article Title: Prophylactic In Vivo Hematopoietic Stem Cell Gene Therapy with an Immune Checkpoint Inhibitor Reverses Tumor Growth in Syngeneic Mouse Tumor Models

doi: 10.1158/0008-5472.can-19-1044

Figure Lengend Snippet: Figure 3. Effect of miR-423-5p target site overexpression on HSPCs. A, Vector structure. HDAd-GFP-miR-423 contains four miR-423-5p target sites in the 30UTR linked to the GFP gene. B, Mouse HSPCs (M; Lin cells from the bone marrow of CD46-transgenic mice) and human HSPCs (Hu; CD34þ cells) were infected with either HDAd-GFP or HDAd-GFP-miR423 at a multiplicity of infection of 500 or 3,000 vp/cell, respectively. Three days later, cell lysates were analyzed by Western blot analysis for CDKN1A. Blots were reprobed with anti-b-actin antibodies to adjust for loading differences. The right panel shows the quantification of CDKN1A signals normalized to b-actin signals. The signals from the corresponding mouse and human HDAd-GFP/mgmt samples were taken as 100%. C, Effect on progenitor colony formation. One day after HDAd infection, mouse Lin cells (2.5 103 cells per 35 mm dish) or human CD34þ cells (3 103 cells/dish) were plated for colony assays. Colonies were counted 12 days later (N ¼ 3; , P < 0.05). Statistical significance was calculated by two-sided Student t test (Microsoft Excel). [In agreement with previous studies (35, 36), infection of HSPCs at relatively high multiplicities of infection slightly reduced the colony forming capacity of HSPCs.] n.s., not significant.

Article Snippet: CDKN1A antibodies (STJ23069, St John's Laboratory) cross-reacted with the mouse and human protein. qRT-PCR for aPD-L1-g 1 mRNA qRT-PCR for aPD-L1-g1 mRNA is described in Supplementary Materials and Methods aPD-L1-g 1 ELISA Recombinant mouse PD-L1 protein (Sino Biological Inc., catalog no. 50010-M08H) at 2 mg/mL were used to coat ELISA plates.

Techniques: Over Expression, Plasmid Preparation, Transgenic Assay, Infection, Western Blot

Journal: British journal of cancer

Article Title: Targeting the actin/tropomyosin cytoskeleton in epithelial ovarian cancer reveals multiple mechanisms of synergy with anti-microtubule agents.

doi: 10.1038/s41416-021-01420-y

Figure Lengend Snippet: Fig. 5 ATM-3507 (ATM) and both anti-microtubule agents, vinorelbine (VNB) and paclitaxel (PTX), enhance G1/S arrest with upregulation of p21 and p27. a The percentage of OVCAR4 cells that underwent different cell fates after completing their first mitosis. At least 85 cells from three independent experiments were analysed for each treatment. b Time cells spent in the next interphase. c Immunoblots and quantification of cyclin D1 and E1 in cell lysates from OVCAR4 cells treated with DMSO, ATM (5 μM), VNB (8 nM), A + V (5 μM ATM + 8 nM VNB), PTX (5 nM) and A + P (5 μM ATM + 5 nM PTX), respectively, for 36 h. Dead and mitotic cells were removed by shake-off. d Immunoblots and quantification of p16, p21 and p27 in cell lysates as in c. Data were collected from at least three independent experiments. Error bars are mean ± S.D.; *p < 0.05; **p < 0.001.

Article Snippet: Antibodies Primary antibodies: mouse anti-Tpm2.1 (CG1; 1:1000); mouse antiTpm3.1 (γ9/d; 1:1000); rabbit anti-Tpm4.1/4.2 (δ9/d; 1:1000); rabbit anti-cyclin E1 (sc-198, Santa Cruz; 1:1000); mouse anti-cyclin D1 (sc-8396, Santa Cruz; 1:500); mouse anti-p27 (3698S, Cell Signaling Technology; 1:1000); mouse anti-p21 (2946T, Cell Signaling Technology; 1:1000); rabbit anti-p16 (ab108349, Abcam; 1:1000); mouse anti-GAPDH (MAB374, Millipore; 1:4000), human anticentromere antibody (15-235-0001, Antibodies Inc.; 1:200); mouse anti-BubR1 (612502, BD Biosciences; 1:500); rabbit anti-BubR1 (phospho S670; ab200062, Abcam; 1:2000); rabbit anti-phosphoHistone H3 (pSer10; Merck; 1:1000).

Techniques: Western Blot